Showing the limitations of available phenotypic assays to detect Burkholderia pseudomallei from clinical specimens in Nigeria

The genus Burkholderia comprises Gram-negative bacteria that are metabolically complex and versatile, often thriving in hostile settings. Burkholderia pseudomallei , the causative agent of melioidosis, is a prominent member of the genus and a clinical pathogen in tropical and sub-tropical regions. This pathogen is well known for its multidrug resistance and possible bioweapon potential. There is currently no report of the pathogen from clinical specimens in Nigeria, which might be due to misdiagnosis with phenotypic assays. This study aims to explore the accuracy of the use of phenotypic assays to diagnose B. pseudomallei in Nigeria. Two hundred and seventeen clinical samples and 28 Gram-negative clinical isolates were collected and analysed using Ashdown’s selective agar and monoclonal antibody-based latex agglutination. Species-level identification was achieved using the analytical profile index (API) 20NE system. The susceptibility of the isolates to nine different antimicrobial agents was determined using the disc diffusion method. A total of seventy-four culture-positive isolates were obtained using Ashdown’s selective agar. Twenty-two of these isolates were believed to be B. pseudomallei through the monoclonal antibody-based latex agglutination test and the API 20NE system subsequently identified 14 isolates as Burkholderia . The predominant Burkholderia species was B. cepacia with an isolation rate of 30.8 % (8/26). No isolate was distinctively identified as B. pseudomallei but five isolates were strongly suspected to be B. pseudomallei with similarity indices ranging from 81.9–91.3 %. Other bacterial species with definitive identity include Aeromonas sp., Sphingomonas sp. and Pseudomonas aeruginosa . The antibiotic susceptibility results revealed an overall resistance to amoxicillin–clavullanic acid of 71.4 %, to cefepime of 33.3 %, to trimethoprim–sulfamethoxazole of 38.1 %, to piperacillin–tazobactam of 33.3 %, to imipenem of 66.7 %, to doxycycline of 57.1% and to ceftazidime of 66.7 %. The highest intermediate resistance was observed for cefepime and piperacillin–tazobactam with a value of 66.7 % each, while there was no intermediate resistance for gentamicin, colistin and imipenem. Our findings, therefore, show that phenotypic assays alone are not sufficient in the diagnosis of melioidosis. Additionally, they provide robust support for present and future decisions to expand diagnostic capability for melioidosis beyond phenotypic assays in low-resource settings.

B. cepacia, B. mallei, B. thailandensis and B. pseudomallei.B. pseudomallei, the causative agent of melioidosis, is an environmental pathogen known to inhabit both soil and water in sub-tropical and tropical regions, especially in Southeast Asia and Northern Australia [5][6][7][8].The potential of B. pseudomallei for use as a bioterrorism agent earns it its classification as a category B agent (second highest priority) by the Centers for Disease Control and Prevention (CDC) [9,10].
B. pseudomallei has been reported in both humans and animals from different parts of Africa [11][12][13].However, to the best of our knowledge, there has been no report of this pathogen from clinical samples in Nigeria despite the country being predicted to have a comparable incidence of melioidosis to that of India, Indonesia and Bangladesh [14].The only report of melioidosis from a Nigerian was in the UK from a Nigerian woman who had visited Nigeria shortly before she was diagnosed with the infection [15].The true source of the infection was not clear and might have suggested that the pathogen was picked up during her visit to Nigeria, especially because the UK's weather conditions do not favour the proliferation of the pathogen.Despite the scarcity of data on the prevalence of the pathogen in clinical samples in Nigeria, the pathogen has been reported in non-clinical samples in the country [16,17].
It can be argued that the lack of data on the clinical occurrence of the pathogens in the country might be due to misdiagnosis.Although standard laboratory media support the growth of B. pseudomallei, specialized agar such as Ashdown's agar, B. cepacia selective agar and B. pseudomallei selective agar are best used for its potential diagnosis [18,19].Unfortunately, these media are not readily available in many diagnostic laboratories in low-resource settings such as Nigeria.Additionally, phenotypic biochemical assays -the most readily available in low-resource settings such as Nigeria -often misidentify B. pseudomallei [20].Collectively, these factors may have contributed to the lack of evidence for the clinical roles of the organism in our hospitals, and the magnitude of its involvement in clinical infections remains to be elucidated.In view of this, we hypothesize that the bacterium may be present and causing infections in our hospitals and we aim to explore the efficiency of the use of phenotypic assays in its identification from clinical specimens.

Study design and specimen collection
This is a cross-sectional study.Two hundred and seventeen clinical specimens from patients and 28 isolates representing a range of non-lactose-fermenting Gram-negative bacilli were examined in this study.All clinical specimens were received and collected by the laboratory department of the participating hospitals and included respiratory secretions, urine, throat swab, sputum and faeces.The sputa had been analysed for tuberculosis bacilli and tested negative using GeneXpert.Both the clinical specimens and isolates were collected over a period of 3 months from Lagos State University Teaching Hospital (LASUTH), Lagos University Teaching Hospital (LUTH) and the Nigerian Institute for Medical Research (NIMR).The age and gender of the patients whose samples or isolates were collected were retrieved from the laboratory records.The study was approved by the institutional Health Research Ethical Committee (reference no.NHREC/19/08/2019B).

Specimen processing and preliminary bacterial identification
The specimens were inoculated onto MacConkey and blood agar for the isolation of non-lactose-fermenting Gram-negative bacilli.Plates were incubated at 37 °C for 24 h [21].The primary identification was made with basic microbiological methods using colony morphology, Gram staining, catalase indole tests, motility tests, oxidase reaction and catalase tests.All isolates were then subcultured on Ashdown's agar and the plates were incubated at 37 °C for 24-48 h.The colony appearance on Ashdown's media was noted.The distinctive purple colour and dry and wrinkled texture of B. pseudomallei colonies can be used to provide a preliminary diagnosis [22].Suspected Burkholderia isolates were subcultured on tryptic soy agar (Himedia) for purity.

Latex agglutination test
A latex agglutination test kit (Mahidol University, Thailand) was used as recommended by the manufacturers.Briefly, approximately 10 µl of the latex reagent containing latex particles coated with monoclonal antibody against B. pseudomallei antigen was pipetted onto a clean glass slide.A sterile inoculating loop was then used to pick the bacterial cell and emulsified in the latex reagent.The slide was subjected to gentle rocking for 2 min, after which the reaction was recorded as either positive (agglutination) or negative (no agglutination).B. thailandensis was used as a negative control.Unfortunately, there was no positive control as standard B. pseudomallei strains were not available and could not be purchased for safety reasons.

Identification using API 20nE biochemical test panel
A cell suspension of a 0.5 McFarland optical density was made with 0.85 % NaCl.An appropriate amount of this suspension was added to each well of the API 20NE strip (BioMerieux, Marcy I, Etoile), some of which were covered with mineral oil.The strips were incubated at 30 °C and read at 24 and 48 h.The results were recorded by visual inspection.Positive or negative reactions were recorded for each test cupule, translated into numerical profiles and interpreted with the APILAB PLUS software package as proposed by the manufacturer.

Statistical analysis
The data were analysed using Microsoft Excel (2016 version) to determine the percentage occurrences/prevalence and the bar charts were made using the ggplot2 package in R (version 4.1.2).

RESuLTS
Cultures of 217 clinical specimens (Data S1) yielded 67 cases of positive growth as non-lactose fermenters on MacConkey agar and typical characteristics on Ashdown's selective agar (Figs 1 and 2).Seven of the 28 Gram negative non-lactose-fermenting bacilli grew on Ashdown's selective agar (ASA).In all, 74 isolates were obtained using Ashdown's selective agar.All culturepositive isolates on Ashdown's media were confirmed to be Gram-negative through Gram staining and microscopy.Seventy of the isolates (70/74; 94.6 %) were rod-shaped and the remaining isolates had the appearance of coccobacilli when viewed under the microscope.Sixty-two of these organisms (62/70; 88.57%) were catalase-positive, while 51 of these organisms (51/70; 72.9 %) were oxidase-positive and only 40 organisms (40/70; 57.1 %) showed positivity in motility testing and showed the presumptive features of Burkholderia.Forty motile isolates with 6 uncertain isolates were subjected to latex agglutination reaction for B. pseudomallei exopolysaccharide; 22 showed agglutination against the monoclonal antibody (Fig. 3).
The majority (15; 68.18 %) of the isolates were from female patients.The largest number of isolates (n=6) was from people within the age range 21-30, followed by people in the 31-40 (n=5) and 41-50 (n=5) age groups.Four samples were from patients in the 51-60 age group.Lastly, one isolate each was obtained from the age groups 11-20 and above the age of 60, while none were from the 0-10 age group (Fig. 4).

DISCuSSIon
In this study, the possible existence of B. pseudomallei in the samples analysed underscores the need to strengthen laboratory capacity for the identification of this pathogen in the clinical microbiology laboratory.Although each of the phenotypic systems utilized was based on the previous evaluation of expressed properties of Burkholderia species, the findings reported here demonstrate that the assays yielded imprecise outcomes for the identification of B. pseudomallei.Ashdown's selective media have been in long-standing use in the literature for the isolation of B. pseudomallei [18].In this study, the overall culture-positive rate for B. pseudomallei-like characteristics was 57.1 %.This is inconsistent with the 5.0 % reported by Schully et al. [28] We observed that the Ashdown's selective medium clearly supported the growth of certain Gram-negative organisms with morphological traits that were comparable to those of B. pseudomallei.These findings are in line with the experiences of other authors [29].Hence, for laboratories unaccustomed to working routinely with these organisms, this may be diagnostically challenging.In this investigation, when compared to a control latex, the antibody-latex suspension recognized 50 %(22/44) of the isolates as positive.In clinical settings, monoclonal antibody-based latex agglutination tests have been shown to be effective in detecting B. pseudomallei [30,31].This latex agglutination test was evaluated against an inclusiveness panel of 77 B. pseudomallei isolates and 76 (98.7 % sensitivity) of these were positive [31].However, members of the B. cepacia complex (Bcc) have been shown to cross-react with B. pseudomallei-specific latex agglutination [32].This observation was confirmed when our isolates were subjected to API 20NE analysis.
Although commercial biochemical panels and nucleic acid amplification techniques have made it possible for diagnostic laboratories to identify most bacterial pathogens with high accuracy and speed, their availability in low-resource settings remains limited, particularly for unusual opportunistic pathogens such as Burkholderia species.Nonetheless, the usefulness of biochemical identification techniques is restricted by the requirement for further tests in order to reliably identify strains to the species level, as well as the nonexistence of some species in the relevant databases [29,33].Whilst some authors have attested to the misidentification tendencies of the API 20NE system [20], it can be constructively suggested that API 20NE has good mechanisms for reliably recognizing B. pseudomallei., [18,30] In several studies, the accuracy of API 20NE for the identification of B. pseudomallei ranged from 37 to 98 % [30,33].In this study, none of the 26 isolates was distinctly identified as B. pseudomallei and 4 isolates appeared to have an alternative similarity index as B. lata, B. gladioli and B. cepacia.These non-B.pseudomallei species can cause melioidosis-like symptoms and share properties with B. pseudomallei at the biochemical level, which could make species-level identification with biochemical panels such as API 20NE challenging.B. gladioli as well as its more distantly related Bcc family, B. cepacia and B. lata, may survive in the same ecological niches as B. pseudomallei.B. gladioli are predominately environmental species and have been implicated in a variety of opportunistic infections, including lung infections [21].Nonetheless, the bulk of our isolates were recognized as B. cepacia, a well-known Bcc member.Given the fact that the global burden of Bcc infections is unknown, members of the group have been documented to infect immunocompromised individuals and drug users [22,34].
In the present paper, Aeromonas occurred at a high proportion, with an identity probability of 82.5-91.2%.Other nonfermentative Gram-negative bacilli (P.aeruginosa and Sphingomonas sp.) were also found, indicating that these bacteria have high survival potential in hospital environments.Due to their perceived ability to develop resistance to several classes of antibiotics, they have become an issue of public health concern, particularly among critically ill patients [27,35,36].The highest frequency of Burkholderia species was found in sputum, with a total percentage of 61.5 % (16/26), in this investigation.This is higher than the findings of Al-Dahash et al. [27], who found 1.6 % B. cepacia among 250 sputa analysed.It is also inconsistent with the findings of Linju et al. [27].Females were found to have a greater number of Burkholderia (62.96 %).This contradicts the results of Linju et al. [37], which showed male dominance, but similar to data from Dizbay et al. [38] on B. cepacia.
This study further buttresses previous reports showing that the use of only phenotypic assays to identify B. pseudomallei in clinical settings might not be sufficient [20,39,40].Greer et al. [39] reported the misidentification of B. pseudmallei as Acinetobacter spp. in their studies.Zong et al. [40] also reported that the VITEK identification system misidentifies B. pseudomallei as B. cepacia.Similarly, Wu et al. [41] reported the misidentification of B. pseudomallei as Aeromonas sobria, Alcaligenes faecalis, Pseudomonas fluorescens and Burkholderia thaillandensis using Vitek 2 Compact, Phoenix, Vitek MS and API 20NE, respectively.Since culturing and phenotypic detection systems such as Vitek and API 20NE are the most common identification assays in many clinical laboratories in Nigeria, it might be difficult to diagnose melioidosis accurately in Nigeria.As a result, there is need to invest in molecular diagnostic tools for effective and accurate clinical diagnosis in the country.
The high prevalence of antimicrobial drug resistance observed among our isolates is alarming and indicates the antibioticresistant nature of Burkholderia, which could make the management of associated infections demanding.The antibiotic susceptibility results revealed that the organisms showed 100 % resistance to gentamicin and colistin.The lowest resistance  was to cefepime and piperacillin-tazobactam.Similar findings were reported by Khosravi et al. [42], who observed that B. pseudomallei from clinical and environmental samples were resistant to multiple antibiotics.On the contrary, Linju et al. [37] described good susceptibility of Burkholderia sp. to ceftazidime, which has been described as the drug of choice for B. pseudomallei infections [29].While Nhung et al. [29] recorded 100 % susceptibility to ceftazidime, imipenem and amoxicillin-clavulanic acid among their B. pseudomallei and B. cepacia isolates, elevated resistance to other antibiotics has been observed by other authors [37].This disparity could be due to the variety of strains collected or perhaps the misuse of antibiotics in Nigeria.
The current research has certain limitations.The first is that the assays were selected and utilized based on the information available in the literature.Secondly, we did not re-evaluate the results for reproducibility for our isolates due to short supply of consumables.In addition, we did not have access to a standard B. pseudomallei strain that could be used as a positive control due to safety reasons.The organism has been classified as a potential bioterrorism organism and is not easily obtained, especially in Nigeria.However, it is noteworthy that the finding suggests the presence of B. pseudomallei in clinical settings and confirms the occurrence of other Burkholderia species.Furthermore, molecular techniques such as 16S rRNA sequencing and whole-genome sequencing would have helped to confirm the identity of the organisms, although we could not use any of these techniques due to resource constraints.Although the clinical implications have not been established, and nor is there a protocol to identify the infection in our setting, scaling up of laboratory capacity is very urgently needed since further studies using genotypic assays will have significant implications for public health.

ConCLuSIon
The limited ability of the phenotypic methods to provide definitive identification of B. pseudomallei was notable.This indicates that misidentification of this organism in hospitals and the associated infections could have been occurring due to under-reporting and poor patient management.The multidrug-resistant nature of the isolates in this study, especially to antibiotics that are often used as drugs of choice for the treatment of bacterial infections, including melioidosis, is a great public health concern.Hence, there is a need for more epidemiological studies on the occurrence and antibiotic resistance of B. pseudomallei in Nigeria, which would include molecular techniques.Efforts should be made to raise awareness and  diagnostic competence, as well as to stimulate case reporting.Clinicians would benefit from having thorough epidemiological data on the organism for the evaluation of melioidosis cases.It is also imperative that clinicians and medical laboratory scientists should be aware of shifting bacterial epidemiology and rising antimicrobial resistance, especially in organisms that are not routinely checked for.This might help health officials and policymakers to implement monitoring, mitigation and preventive efforts for curtailing antimicrobial resistance.
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Find out more and submit your article at microbiologyresearch.org birds (15), freshwater fishes ( 16) and hospital effluents ( 17)", but reference 17 is about the misidentification of different species of bacteria, and it is in fact reference 8 which talks about detection in hospital effluents.Another example is reference 1 which is a review article discussing immune evasion mechanisms but it is being used as a reference for the statement "The Gram-negative bacteria, Burkholderia, are ecologically diverse genus with over 70 species whose heterogeneousness has led to multiple taxonomic changes over time 1, 2".This is not an appropriate reference for that statement.Please carefully check that all references are backing up the statements being made, and are the appropriate primary sources of information.Other minor changes required; Fig 5 legend-recurrence should be occurrence Fig 3-please rotate the X axis labels to be horizontal Ashdown should be Ashdown's throughout the manuscript Lastly, I think the data provided in the supplementary excel sheet could be made more useful if the phenotypic test results (catalase, oxidase, motility, agglutination) for each of the strains was added to sheet "sample info of identified strains" so the reader could see what combination of test results make up the diagnostic profile of each species.

Response to revision
Dear Editor, We have revised the manuscript as requested by the reviewers.Below are some of the revisions made; • The title of the article has been changed based on reviewers' recommendations -line 2. The title has been changed to "Showing the limitations of available phenotypic assays to detect B. pseudomalleifrom clinical specimens in Nigeria".
• lines 147-149 -The "n" has been changed to real numbers.
• A flow chart has been added to show the phenotypic assay steps and the number of samples tested at each step -line 158.
• The sample numbers have been reconciled and more information has been provided in the supplementary file.For instance the number of samples positive to agglutination has been changed from 26 to 22 in the abstract in line 36.The number of clinical specimens in line 166-168 has been reconciled and corrected as appropriate.
• A new paragraph providing evidence to support the inaccuracy in the use of phenotypic assays to identify B. pseudomalleilines 278-287.
• Lines 147 to 149 -the origin of the 26 isolates on which API 20NE identification was done has been added to the supplementary data.
• Line 149 -The isolates were confirmed via Gram staining and microscopy, this has been added to the sentence in line 150.
• Line 173 -the number (26) of isolates tested with API20NE has been added to the supplementary data as advised by reviewer 2.
• Figures 4 and 5 (previously figures 3 and 4) have also been changed to new figures.Figure 4 has been changed to a new barplot showing the numbers for males and females side by side as against the stacked bar plot used previously (Line 177).
• Line 132-134 -Three references where similar antibiotics were used for AST of B. pseudomalleihas been added (can be found in lines 136-137) • Other minor revisions have been made as suggested by the reviewers.For instance, the misspelt pseudomallei in line 24 has been spelt correctly, the statement referring to heatmaps in line 141 has been removed to reflect the removed heatmap (as advised by the editor when the manuscript was first submitted).Lastly, all data sheets in the supplementary data has been named appropriately.
Thank you for the revision Comments: Thank you to both reviewers for their thorough reports on this manuscript.Please revise your manuscript in line with these comments, including paying close attention to discrepancies in number of samples reported at different points throughout the manuscript.I agree with both reviewers that a title change would be beneficial to reflect the findings of the manuscript, rather than a description of the work that has been carried out.Reviewer 2's suggestion to add a flow chart describing the number of samples at each diagnostic stage would help to make clearer the testing strategy.Comments: This article is a good reminder that phenotypic assays are still a unique resource for some countries.Thus, it is important to improve identification tests when resources are limited especially since sequencing is routinely used for isolate identification in the majority of countries.The authors have proven that existing phenotypic assays fail to identify closed-related bacterial species within the Burkholderia genus.1. Methodological rigour, reproducibility and availability of underlying data The article comprises minor mistakes, such as spelling, missing information, and mistakes in reported numbers between the text and the figures.Most of the supplementary data are missing.Adding them will add strength to the article.I believe that the study does not request any complementary experiments.However, I have noted that the article lacks linking bacterial isolates with their original origin (provided in sheet 1 in Excel supplementary data) and their phenotypic characterization.I believe that the phenotypic assays used in this study are specific for Burkholderia identification.However, the method part is missing information about protocol and references for most phenotypic assays.Without this information, it will be difficult to reproduce the experiments.2. Presentation of results In my opinion, provided figures are sufficient for the conclusion of the study.I have made some suggestions to improve the figures.3. How the style and organization of the paper communicates and represents key findings The article follows a logical process to identify bacterial isolates based on phenotypic assays.4. Literature analysis or discussion The authors have discussed their results with the literature and have identified the limitations of their study including the confirmation of the species of the identified Burkholderia isolates and the lack of repeat experiments due to low resources.I believe that this PCR or sequencing verification and repeating the identification process would have added strength to the study.However, all phenotypic assays direct to the same conclusion: they fail identifying bacterial species within a bacterial genus. 5. Any other relevant comments Please find the comments: Title: I would suggest changing the title to represent the main conclusion of the study; showing the limitations of available phenotypic assays to detect B. pseudomallei in Nigeria.Line 24: misspelling of "pseudomallei" Line 104-106: Please provide more information and references about protocols and replicates used for all phenotypic assays.Could you also provide pictures or data for the colony morphotypes for isolates?Line 132-134: Please provide references to support the antibiotic quantities used for the antimicrobial resistance assay.Line 138: the dot at the end of the sentence is missing.Line 141: There are no heatmaps in the manuscript.3: My first understanding is the graph is a stacked barplot.However, from the description in the text at line 166, it seems that the female columns are in the background of the male columns.I would suggest either separating female and male into two columns side by side with the actual y-axis or using a stacked barplot where one column is split between male/female with a total number of both sexes.Figure 4 B. cenocepacia isolates was also reported (Cross-reactivity of latex agglutination assay complicates the identification of Burkholderia pseudomallei from soil -PubMed ( nih.gov)).The results of identification were inconclusive, maybe due to lack of testing proficiency (e.g.not using correct controls).There were several isolates identified as Burkholderia which maybe were or maybe were not B. pseudomallei.Antimicrobial susceptibility of these isolates was tested by disc diffusion, which is one of EUCAST recommended methods for this bacterium.The high levels of resistance to clinically important antimicrobials for B. pseudomallei were found (ceftazidime, imipenem), but significance of this finding is not clear as the isolates might not be B. pseudomallei.Comments.1.
From the start, the title is grammatically incorrect and misleading: "specimen" should be plural "specimens" ; "Ashdown's" not "Ashdown" is the correct name for the agar, but it is not needed in the title because AST is not be performed on this agar; most importantly, the authors state (lines 37-38) "No isolate was distinctively identified as B. pseudomallei", then why "Burkholderia pseudomallei" is in the title? 2. References do not cite the original work and/or do not correspond to the text at all.The example of completely wrong use is the reference is #6 which should refer to "B. pseudomallei designated a bioterrorism agent" but it is "Adebowale O, Adeyemo O. Characterization of bacterium types isolated from commercial laying hen farms in Ogun State Nigeria.Rev élev méd vét pays trop.2018; 71(3):137-141."References 8,9 supposed to list endemic areas, but they are susceptibility papers from Nigeria.#14 is not 2011 Salam paper.All references need to be verified.3.
Methods are mainly well written and appropriate.Statistical analysis method needs explanation on which data were analyzed.Was it AST data?Results: 4. not clear what is "n" is in lines 147-149. 5.
Line 150: delete "and" 6.For latex agglutination: in the Abstract, 26 isolates were reported positive by latex agglutination (line 34), in Results -22 isolates were reported positive (line 155).Which one is correct?Which isolates were tested by latex agglutination?From Suppl Material, 45 isolates were tested but not clear, how they were selected for the test.From Abstract, it sounds that 67 isolates strains that grew on Ashdown's from 217 clinical specimens were tested and none of the 28 Gram-negative clinical isolates were tested.This needs verification and explanation.7  The current use of a heat map means that reviewers cannot see from this figure how many samples fall into each category, and are left guessing as to how many samples were positive and negative for each test type.I know the numbers are stated in the text, but the graph needs to be useful in its own right.Similarly for figure 3 a histogram should be used to display this age category data, not a heat map.On figure 4, it is not possible to see the frequency data as it is hidden behind the % occurrence bars.Please re-graph the data in the manuscript so that we may consider it for peer review.

Fig. 1 .
Fig. 1.Flow chart showing the phenotypic identification steps and number of samples tested at each stage.

Fig. 3 .
Fig. 3. Biochemical assays for characterization of bacterial pathogens from Ashdown's selective agar.The isolates are grouped into positive and negative.

Fig. 4 .Fig. 5 .
Fig. 4. Gender and age distribution of patients for clinical specimens from which isolates originated.

Reviewer 2
recommendation and comments https://doi.org/10.1099/acmi.0.000604.v2.1 © 2023 Coulon P.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License.Pauline Coulon; NA, AUSTRALIA Date report received: 23 June 2023 Recommendation: Minor Amendment

Figure 2 Figure 3 Figure 4
Figure 2 has been changed to bar charts Figure 3 has also been changed to a bar chart Figure 4 has been modified to allow the frequency values show.Thank you

Table 1 .
Antimicrobial susceptibility profiles of Burkholderia species isolated from clinical specimens

Table 2 .
Multidrug resistance profiles for Burkholderia isolates Lines 144-146: Sheet 1 from the supplementary data Excel file records 218 patients/clinical specimens, the text says 217.The result of growth on both MacConkey Agar and Ashdown medium should be linked to the clinical specimen in supplementary data.Lines 147 to 149: The origin of samples should be added as supplementary data.Line 149: Please specify how the isolates have been confirmed to be Gram-negative.Line 149-156: Please add the positive/negative results for each performed test in supplementary data.Line 166-168: The numbers of clinical specimens isolated from patients ranked by age do not match with Figure3.For example, 7 isolates belong to the age 31-40 group in the text while the graph only shows five females + one male if my understanding is correct.Line 173: Please specify the total number of isolates that have been tested by API 20NE and the raw data in supplementary data.I am not sure what is "(99.9%ID,".Line 175: Supplementary data are missing.Line 179: I believe that "Figure3" should be Figure4.Line 188: Please justify the choice of the antimicrobials that have been tested.Line 203: Please provide details for the characterization by including the range of zones of inhibition that define Susceptible, Intermediate and Resistance phenotypes.Line 195: I would suggest adding the abbreviation used for each antibiotic in the paragraph.Line 255: "B.cepacia", B is missing italic font.Figure2: I would suggest adding a flowchart describing the results (from 217/218 clinical specimens to 26 API-tested isolates) with the barplot for each test to prove the results.Figure : I would suggest adding two y-axes for the graph with names.The supplementary data Excel file name is "Burlkhoderia" instead of Burkholderia.The First sheet is not named.Supplementary data should be named by tables and figures and reported by table and figure numbers in the text.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License.Diagnosis is critical to successfully treat Burkholderia pseudomallei, the cause of melioidosis, underlying importance of this study.Here, the authors tested 217 clinical samples and 28 gram-negative purified bacterial isolates in attempt to identify B. pseudomallei.API 20NE and latex agglutination tests which were used by authors are accurate for identification of B. pseudomallei (Accuracy of Burkholderia pseudomallei Identification Using the API 20NE System and a Latex Agglutination Test -PMC ( nih.gov)) but cross-reactivity of latex agglutination tests for melioidosis with B. territorii, B. pseudomultivorans, B. multivorans and

Author response to reviewers to Version 1
. Line 179 -should this be Figure4?9.Figures: Legends need more details.The font is too small in the figures.10.Table1and AST results.Is it possible to abbreviate antimicrobial names according to -Abbreviations and Conventions ( asm.org) ?Please rate the manuscript for methodological rigour SatisfactoryPlease rate the quality of the presentation and structure of the manuscript PoorTo what extent are the conclusions supported by the data?Partially supportDo you have any concerns of possible image manipulation, plagiarism or any other unethical practices?NoIs there a potential financial or other conflict of interest between yourself and the author(s)?NoIf this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?Yes Revision response Overall, figures 2, 3, and 4 have all been modified based on the editor's suggestions.
. Which isolates were analyzed by API 20NE?Did the authors find 26 or 8 B. cepacia?-line 173.8